Lujian Liao

The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal Secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

βCaMKII in Lateral Habenula Mediates Core Symptoms of Depression

Depression and the Habenula The lateral habenula (LHb) appears to have a role in depression. However, the underlying mechanisms are poorly understood, and by using multiple rodent models of depression, Li et al. (p. 1016) identified a signaling pathway and associated neuronal adaptations in which the enzyme βCaMKII was selectively up-regulated in the LHb. Manipulations that enhanced βCaMKII levels increased depression-related phenotypes, and RNA interference of CaMKIIb blunted depression. Enhanced βCaMKII levels in the habenula promoted excitatory synaptic transmission on these neurons and increased action potential firing mediated by an up-regulation of a specific subtype of glutamate receptors. In a rodent model of depression, the increase of a protein kinase is accompanied by an up-regulation of GluR1 receptors. The lateral habenula (LHb) has recently emerged as a key brain region in the pathophysiology of depression. However, the molecular mechanism by which LHb becomes hyperactive in depression remains unknown. Through a quantitative proteomic screen, we found that expression of the β form of calcium/calmodulin-dependent protein kinase type II (βCaMΚΙΙ) was significantly up-regulated in the LHb of animal models of depression and down-regulated by antidepressants. Increasing β-, but not α-, CaMKII in the LHb strongly enhanced the synaptic efficacy and spike output of LHb neurons and was sufficient to produce profound depressive symptoms, including anhedonia and behavioral despair. Down-regulation of βCaMKII levels, blocking its activity or its target molecule the glutamate receptor GluR1 reversed the depressive symptoms. These results identify βCaMKII as a powerful regulator of LHb neuron function and a key molecular determinant of depression.

Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice

Fragile X syndrome (FXS) is a common inherited form of mental retardation that is caused, in the vast majority of cases, by the transcriptional silencing of a single gene, fmr1. The encoded protein, FMRP, regulates mRNA translation in neuronal dendrites, and it is thought that changes in translation-dependent forms of synaptic plasticity lead to many symptoms of FXS. However, little is known about the potentially extensive changes in synaptic protein content that accompany loss of FMRP. Here, we describe the development of a high-throughput quantitative proteomic method to identify differences in synaptic protein expression between wild-type and fmr1−/− mouse cortical neurons. The method is based on stable isotope labeling by amino acids in cell culture (SILAC), which has been used to characterize differentially expressed proteins in dividing cells, but not in terminally differentiated cells because of reduced labeling efficiency. To address the issue of incomplete labeling, we developed a mathematical method to normalize protein ratios relative to a reference based on the labeling efficiency. Using this approach, in conjunction with multidimensional protein identification technology (MudPIT), we identified >100 proteins that are up- or down-regulated. These proteins fall into a variety of functional categories, including those regulating synaptic structure, neurotransmission, dendritic mRNA transport, and several proteins implicated in epilepsy and autism, two endophenotypes of FXS. These studies provide insights into the potential origins of synaptic abnormalities in FXS and a demonstration of a methodology that can be used to explore neuronal protein changes in neurological disorders.

HDAC4 governs a transcriptional program essential for synaptic plasticity and memory.

Neuronal activity influences genes involved in circuit development and information processing. However, the molecular basis of this process remains poorly understood. We found that HDAC4, a histone deacetylase that shuttles between the nucleus and cytoplasm, controls a transcriptional program essential for synaptic plasticity and memory. The nuclear import of HDAC4 and its association with chromatin is negatively regulated by NMDA receptors. In the nucleus, HDAC4 represses genes encoding constituents of central synapses, thereby affecting synaptic architecture and strength. Furthermore, we show that a truncated form of HDAC4 encoded by an allele associated with mental retardation is a gain-of-function nuclear repressor that abolishes transcription and synaptic transmission despite the loss of the deacetylase domain. Accordingly, mice carrying a mutant that mimics this allele exhibit deficits in neurotransmission, spatial learning, and memory. These studies elucidate a mechanism of experience-dependent plasticity and define the biological role of HDAC4 in the brain.

Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3

ABSTRACT Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

K33-Linked Polyubiquitination of T Cell Receptor-ζ Regulates Proteolysis-Independent T Cell Signaling

Tagging the cell surface receptor with ubiquitin is believed to provide a signal for the endocytic pathway. E3 ubiquitin ligases such as Cbl-b and Itch have been implicated in T cell activation and tolerance induction. However, the underlying mechanisms remain unclear. We describe that in mice deficient in the E3 ubiquitin ligases Cbl-b and Itch, T cell activation was augmented, accompanied by spontaneous autoimmunity. The double-mutant T cells exhibited increased phosphorylation of the T cell receptor-zeta (TCR-zeta) chain, whereas the endocytosis and stability of the TCR complex were not affected. TCR-zeta was polyubiquitinated via a K33-linkage, which affected its phosphorylation and association with the zeta chain-associated protein kinase Zap-70. The juxtamembrane K54 residue in TCR-zeta was identified to be a primary ubiquitin conjugation site, whose mutation increased its phosphorylation and association of TCR-zeta and Zap-70. Thus, the present study reveals unconventional K33-linked polyubiquitination in nonproteolytic regulation of cell-surface-receptor-mediated signal transduction.

Proteomic identification of novel proteins associated with Lewy bodies.

The manifestation of Lewy bodies (LB) in the brain is a hallmark of Parkinsons disease. Here, we present a comprehensive analysis of protein elements in Lewy bodies by comparative mass spectrometry. Cortical LB inclusions were enriched by sucrose gradient centrifugation from postmortem brains, and a negative control sample was prepared from specimen without LB pathology. Whereas approximately 550 proteins were identified in the LB-enriched sample by mass spectrometry, quantitative comparison with the control sample revealed that approximately 40 proteins were co-enriched with alpha-synuclein, the major component in Lewy bodies. As expected, the list of proteins included previously reported constituents, such as those involved in protein folding, membrane trafficking and oxidative stress. More interestingly, we discovered in the LB-enriched sample several kinases (MAPKK1/MEK1, protein kinase C, and doublecortin-like kinase), a novel deubiquitinating enzyme (otubain 1), and numerous ubiquitin ligases (KPC and SCF). The proteomic studies provide enzyme candidates to investigate the regulation of alpha-synuclein and/or other LB proteins, which may contribute to the formation of Lewy bodies and the toxicity of alpha-synuclein in the related neurodegenerative disorders.

Neuronal Morphogenesis Is Regulated by the Interplay between Cyclin-Dependent Kinase 5 and the Ubiquitin Ligase Mind Bomb 1

Neuronal communication requires the coordinated assembly of polarized structures including axons, dendrites, and synapses. Here, we report the identification of a ubiquitin ligase mind bomb 1 (Mib1) in the postsynaptic density and the characterization of its role in neuronal morphogenesis. Expression of Mib1 inhibits neurite outgrowth in cell culture and its gene deletion enhances synaptic growth at the neuromuscular junction in Drosophila. The analysis of Mib1 interactome by mass spectrometry revealed that Mib1 primarily interacts with membrane trafficking proteins [e.g., EEA1 (early endosomal antigen 1), Rab11-interacting proteins, and SNAP25 (synaptosomal-associated protein of 25 kDa)-like protein] and cell adhesion components (e.g., catenin, coronin, dystrobrevin, and syndecan), consistent with its previously reported function in protein sorting. More interestingly, Mib1 is associated with deubiquitinating enzymes, BRCC36 and the mammalian ortholog of fat facets, and a number of kinases, such as casein kinase II, MARK (microtubule affinity regulating kinase)/PAR1, and cyclin-dependent kinase 5 (CDK5). Further characterization of the Mib1-CDK5 interaction indicated that the N-terminal domain of Mib1 directly binds to the regulatory subunit p35 of the CDK5 complex. In cell culture, Mib1 induces the relocalization of p35/CDK5 without affecting its degradation. Surprisingly, p35/CDK5 downregulates the protein level of Mib1 by its kinase activity, and completely rescues the Mib1-induced inhibitory effect on neurite morphology. p35/CDK5 also genetically interacts with Mib1 in the fly according to the rough-eye phenotype. The data strongly support that the negative interplay between Mib1 and p35/CDK5 may integrate the activities of multiple pathways during neuronal development.

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology.

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

Quantitative analysis of brain nuclear phosphoproteins identifies developmentally regulated phosphorylation events

Protein phosphorylation is a globally adopted and tightly controlled post-translational modification, and represents one of the most important molecular switching mechanisms that govern the entire spectrum of biological processes. In the central nervous system, it has been demonstrated that phosphorylation of key proteins mediating chromatin remodeling and gene transcription plays an important role controlling brain development, synaptogenesis, learning and memory. Many studies have focused on large scale identification of phosphopeptides in brain tissue. These studies have identified phosphorylation site specific motifs useful for predicting protein kinase substrates. In this study, we applied a previously developed quantitative approach, stable isotope labeling of amino acids in mammals (SILAM), to quantify changes in the phosphorylation of nuclear proteins between a postnatal day one (p1) and a p45 rat brain cortex. Using a 15N labeled rat brain as an internal standard, we quantified 705 phosphopeptides in the p1 cortex and 1477 phosphopeptides in the p45 cortex, which translates to 380 and 585 phosphoproteins in p1 and p45 cortex, respectively. Bioinformatic analysis of the differentially modified phosphoproteins revealed that phosphorylation is upregulated on multiple components of chromatin remodeling complexes in the p1 cortex. Taken together, we demonstrated for the first time the usefulness of employing stable isotope labeled rat tissue for global quantitative phosphorylation analysis.